CN115475250B - Drug-loaded exosome targeting hepatic stellate cells and inhibiting activation thereof, and preparation and application thereof - Google Patents
Drug-loaded exosome targeting hepatic stellate cells and inhibiting activation thereof, and preparation and application thereof Download PDFInfo
- Publication number
- CN115475250B CN115475250B CN202210578625.0A CN202210578625A CN115475250B CN 115475250 B CN115475250 B CN 115475250B CN 202210578625 A CN202210578625 A CN 202210578625A CN 115475250 B CN115475250 B CN 115475250B
- Authority
- CN
- China
- Prior art keywords
- hepatic stellate
- drug
- exosome
- pancreatic cancer
- cells
- 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.)
- Active
Links
- 210000004024 hepatic stellate cell Anatomy 0.000 title claims abstract description 85
- 210000001808 exosome Anatomy 0.000 title claims abstract description 80
- 239000003814 drug Substances 0.000 title claims abstract description 50
- 229940079593 drug Drugs 0.000 title claims abstract description 45
- 230000004913 activation Effects 0.000 title claims abstract description 23
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 21
- 230000008685 targeting Effects 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title description 4
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims abstract description 26
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims abstract description 26
- 201000002528 pancreatic cancer Diseases 0.000 claims abstract description 26
- 208000008443 pancreatic carcinoma Diseases 0.000 claims abstract description 26
- 210000004027 cell Anatomy 0.000 claims abstract description 24
- 230000020411 cell activation Effects 0.000 claims abstract description 9
- 230000002300 anti-fibrosis Effects 0.000 claims abstract description 4
- ISWRGOKTTBVCFA-UHFFFAOYSA-N pirfenidone Chemical compound C1=C(C)C=CC(=O)N1C1=CC=CC=C1 ISWRGOKTTBVCFA-UHFFFAOYSA-N 0.000 claims description 17
- 229960003073 pirfenidone Drugs 0.000 claims description 16
- 238000000108 ultra-filtration Methods 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 4
- 239000000523 sample Substances 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 238000000034 method Methods 0.000 claims description 3
- 238000002604 ultrasonography Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 238000001085 differential centrifugation Methods 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 238000010257 thawing Methods 0.000 claims description 2
- 206010027457 Metastases to liver Diseases 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 206010027476 Metastases Diseases 0.000 abstract description 17
- 206010028980 Neoplasm Diseases 0.000 abstract description 17
- 230000009401 metastasis Effects 0.000 abstract description 15
- 206010016654 Fibrosis Diseases 0.000 abstract description 9
- 230000004761 fibrosis Effects 0.000 abstract description 9
- 210000004185 liver Anatomy 0.000 abstract description 9
- 230000015572 biosynthetic process Effects 0.000 abstract description 7
- 238000001262 western blot Methods 0.000 abstract description 6
- 230000035755 proliferation Effects 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 4
- 238000000338 in vitro Methods 0.000 abstract description 3
- 238000004393 prognosis Methods 0.000 abstract description 3
- 230000002452 interceptive effect Effects 0.000 abstract description 2
- 210000003712 lysosome Anatomy 0.000 description 7
- 230000001868 lysosomic effect Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 4
- 230000002440 hepatic effect Effects 0.000 description 4
- 230000001394 metastastic effect Effects 0.000 description 4
- 206010061289 metastatic neoplasm Diseases 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 2
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 2
- 208000005443 Circulating Neoplastic Cells Diseases 0.000 description 2
- 102000016359 Fibronectins Human genes 0.000 description 2
- 108010067306 Fibronectins Proteins 0.000 description 2
- 102000003745 Hepatocyte Growth Factor Human genes 0.000 description 2
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 2
- 201000009794 Idiopathic Pulmonary Fibrosis Diseases 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- VBVAVBCYMYWNOU-UHFFFAOYSA-N coumarin 6 Chemical compound C1=CC=C2SC(C3=CC4=CC=C(C=C4OC3=O)N(CC)CC)=NC2=C1 VBVAVBCYMYWNOU-UHFFFAOYSA-N 0.000 description 2
- 230000003176 fibrotic effect Effects 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 208000036971 interstitial lung disease 2 Diseases 0.000 description 2
- 201000008129 pancreatic ductal adenocarcinoma Diseases 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 238000000035 BCA protein assay Methods 0.000 description 1
- 102100025222 CD63 antigen Human genes 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 206010019695 Hepatic neoplasm Diseases 0.000 description 1
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 description 1
- 101000613251 Homo sapiens Tumor susceptibility gene 101 protein Proteins 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 102100040879 Tumor susceptibility gene 101 protein Human genes 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000003510 anti-fibrotic effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008045 co-localization Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 1
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000003921 particle size analysis Methods 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- 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
- C12N5/0676—Pancreatic 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
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Oncology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Botany (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention provides a drug-loaded exosome for targeting hepatic stellate cells and inhibiting activation of the hepatic stellate cells, which is obtained by introducing an anti-fibrosis drug for inhibiting activation of the hepatic stellate cells into an exosome with hepatic stellate cell targeting, wherein the exosome with hepatic stellate cell targeting is derived from a pancreatic cancer cell line. The hepatic stellate cell targeted drug-loaded exosome provided by the invention has expected safe treatment effect on tumors and in-vitro proliferation experiments of hepatic stellate cells, and Western blot experiments on hepatic stellate cell stem prognosis also show that the hepatic stellate cell targeted drug-loaded exosome can inhibit hepatic stellate cell activation. The hepatic stellate cell targeted drug-loaded exosome is used for interfering with the formation of the pre-fibrosis metastasis niche, so that possibility is provided for inhibiting the liver metastasis of pancreatic cancer, and a hint is provided for the treatment of other tumor metastases.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a drug-loaded exosome which targets hepatic stellate cells and inhibits activation of hepatic stellate cells, and preparation and application thereof.
Background
Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most invasive cancers accepted, with a 5-year survival rate of only about 10% among all malignant tumors, with an extremely high degree of malignancy being associated with a range of factors, probably the most prominent reason for its high metastatic potential. Among all organs susceptible to distant metastasis, the most common distant metastasis site is the liver (76-80%). In the "seed and soil" theory of tumor metastasis, the interaction between cancer cells and the remote microenvironment optimizes the potential for engraftment of circulating tumor cells. Cancer cells in primary tumors induce, in advance of their spread, in secondary organs, the formation of microenvironments that favour their metastatic invasion, i.e. pre-metastatic niches.
The occurrence of metastasis is largely determined by the extracellular vesicles (EV, including exosomes) of primary tumor origin and the local interstitial microenvironment of the metastatic organ. Exosomes are approximately 40-160 nm in size, originate from the exosomes, and are involved in a variety of physiological and pathological processes in the body including tumor metastasis. For example, in vitro transport of EGFR from gastric cancer cells to hepatic stellate cells promotes liver-specific metastasis by enhancing HGF (hepatocyte growth factor) signaling in the liver. The occurrence of microenvironment fibrosis is masked, and tumor cells in the circulation can be activated by adhesion to the microenvironment, thereby promoting proliferation. Thus, there is an urgent need for drug-loaded exosomes that target hepatic stellate cells and inhibit their activation.
Disclosure of Invention
Research proves that exosomes derived from pancreatic primary tumors can be taken up by Hepatic Stellate Cells (HSCs), and the generation of alpha-SMA and fibronectin in the HSCs is up-regulated, so that a fibrous microenvironment is formed in the liver, and the exosomes derived from pancreatic cancer primary tumors can overcome the concealment of the occurrence of the microenvironment fibrosis and have the potential of accurately targeting to the hepatic stellate cells. The invention aims to provide a drug-loaded exosome which targets hepatic stellate cells and inhibits activation of the hepatic stellate cells, and preparation and application of the drug-loaded exosome, so as to intervene in the generation of fibrosis microenvironment mainly caused by the activation of the hepatic stellate cells, thereby inhibiting the liver metastasis of pancreatic cancer and solving the problems in the background.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a drug-loaded exosome that targets and inhibits activation of hepatic stellate cells, the drug-loaded exosome being obtained by introducing an anti-fibrotic drug for inhibiting activation of hepatic stellate cells into an exosome having hepatic stellate cell targeting derived from a pancreatic cancer cell line.
In order to optimize the technical scheme, the specific measures adopted further comprise:
further, sources of the pancreatic cancer cell line include, but are not limited to, human pancreatic cancer cell line MIA-Paca2, mouse pancreatic cancer cell line mT7.
Further, the anti-fibrosis drugs used to inhibit hepatic stellate cell activation include, but are not limited to, pirfenidone.
Further, the drug-loaded exosomes targeting hepatic stellate cells and inhibiting their activation are prepared by the following method:
s1, culturing pancreatic cancer cells, collecting a conditional culture medium, and obtaining exosomes derived from the pancreatic cancer cells after differential centrifugation, ultrafiltration concentration, super-separation or exosome extraction reagent incubation;
s2, after the exosome precipitation is resuspended by PBS, a pirfenidone solution is added, and the target product is obtained after circulating freeze thawing, probe ultrasound, water bath balancing and ultrafiltration of superfluous free medicine.
Further, the drug-loaded exosomes targeting hepatic stellate cells and inhibiting activation thereof are used for preparing drugs for treating pancreatic cancer liver metastasis; specifically, the drug-loaded exosome can inhibit tumor liver metastasis by inhibiting the formation of a fibrosis microenvironment, pancreatic cancer cell-derived exosome can actively target hepatic stellate cells, and the drug-loaded exosome can inhibit the activation of hepatic stellate cells after carrying pirfenidone.
The beneficial effects of the invention are as follows: the hepatic stellate cell targeted drug-carrying exosome provided by the invention targets and transports the fibrosis drug to hepatic stellate cells; wherein, the exosomes are derived from pancreatic cancer cell lines, and have the advantages of good compatibility, permeability, natural stability, low immunogenicity, toxicity and the like; the fibrosis medicine is Pirfenidone (PF), which is a pharmacological compound with therapeutic effect on Idiopathic Pulmonary Fibrosis (IPF) and has the effect of regulating fibrotic growth factors, thereby weakening proliferation of fibroblasts, synthesis of collagen and fibronectin, and deposition of extracellular matrix; after a series of membrane rupture and reconstruction are carried out on the exosome of the pancreatic cancer cell line, the composition of active ingredients for promoting the tumor progress is changed, and the biological safety is ensured; after the fibrotic drug is transported to hepatic stellate cells, the formation of niches suitable for the colonization of circulating tumor cells is intervened, thereby inhibiting liver metastasis of pancreatic cancer. The in vitro proliferation experiment obtains the expected safe treatment effect, and the Western blot experiment on hepatic stellate cell stem prognosis also shows that the hepatic stellate cell stem prognosis can inhibit hepatic stellate cell activation. The hepatic stellate cell targeted drug-loaded exosome is used for interfering with the formation of the pre-fibrosis metastasis niche, so that possibility is provided for inhibiting the liver metastasis of pancreatic cancer, and a hint is provided for the treatment of other tumor metastases.
Drawings
FIG. 1 is a particle size detection and transmission electron microscopy image of drug-loaded exosomes targeting hepatic stellate cells and inhibiting their activation;
FIG. 2 is a graph showing the results of lysosome escape of drug-loaded exosomes in hepatic stellate cells targeted to and inhibited from activation by hepatic stellate cells;
FIG. 3 is a graph showing drug-loaded exosomes uptake within hepatic stellate cells targeted to and inhibited from activation by hepatic stellate cells;
FIG. 4 is a Western blot detection of drug-loaded exosomes activated marker proteins targeting hepatic stellate cells and inhibiting activation thereof;
FIG. 5 is a Western blot assay with hepatic stellate cell activation inhibited;
FIG. 6 is a CCK8 assay of drug-loaded exosomes targeting and inhibiting activation of hepatic stellate cells in terms of tumor cell, hepatic stellate cell proliferation safety.
Detailed Description
EXAMPLE 1 exosome isolation and drug delivery
Exosome MIA-Paca-2 secreted by human pancreatic cancer cells was extracted and collected using an exosome extraction kit (Henan Bebei technologies).
MIA-Paca-2 cell culture solution is cultured by exosome-free serum and DMEM culture medium; centrifuging for 5min under the centrifugal force of 300g, and collecting supernatant; centrifuging for 20min under a centrifugal force of 2000g, and collecting supernatant; centrifuging under 12000g centrifugal force for 35min, collecting supernatant, and discarding cell debris and precipitate; centrifuging the centrifuged cell culture solution with a 100kD ultrafiltration tube under the centrifugal force of 2000g for 15min, taking the upper chamber liquid, filtering with a 0.22 μm filter membrane, adding 1/3 volume of exosome extraction reagent, mixing uniformly, placing in a shaker for incubation at 4 ℃ for 1h, centrifuging for 20min under the centrifugal force of 12000rpm, re-suspending and collecting the precipitate with sterile PBS, and preserving at 4 ℃ for a short period of time to obtain hepatic stellate cell targeted exosomes. The BCA protein assay coarsely measured the concentration of the exosome protein at 10. Mu.g/. Mu.L.
Adding pirfenidone solution (10 mg/mL, solvent PBS) into the obtained exosome solution according to the volume ratio of 1:1, freezing for 5min with liquid nitrogen, balancing for 10min at room temperature, and repeating for 2 times; ultrasonic treatment of probe under ice bath for 5min (ultrasonic treatment for 3s at intervals of 2s, maximum temperature of 30deg.C and power of 9%), balancing at room temperature for 10min, and repeating for 2 times; water bath balance at 37 ℃ for 1h to restore the stability of the membrane; adding a 100kD ultrafiltration tube, centrifuging for 5min under 2000g centrifugal force, adding PBS for washing, centrifuging for 5min under 2000g centrifugal force, and collecting upper chamber liquid to obtain hepatic stellate cell targeted drug-carrying exosome. After dilution with DMSO and ultrasound with a probe, the absorbance was measured at 310nm using an ultraviolet spectrophotometer, and the pirfenidone concentration was calculated from the standard curve to 3-4. Mu.g/. Mu.L.
And performing Western blot, electron microscope and particle size detection of marker proteins on the hepatic stellate cell targeted exosomes and the hepatic stellate cell targeted drug-loaded exosomes which are separated and collected. Western blot results show that the exosome markers CD63 and TSG101 are highly expressed; the electron microscope result shows that both the hepatic stellate cell targeted exosome and the hepatic stellate cell targeted drug-carrying exosome can see double concave circular disc vesicles; particle size analysis detected particle sizes in the range of 60-120nm, and the specific results are shown in FIG. 1.
Example 2 lysosome escape procedure for hepatic stellate cell-targeted exosomes
The nanoscale medicine enters cells often in endocytosis mode, is taken up by lysosomes, and can play a role in cytoplasm after being released from the lysosomes. The hepatic stellate cell targeted exosomes obtained in example 1 were entrapped with the fluorescent dye coumarin 6 (C6) in the same manner, were treated by adding to hepatic stellate cells Lx2, and were subjected to lysosome tracer reagent addition at 0.5,1,2h, respectively, and the positional relationship between the hepatic stellate cell targeted exosomes (green) and lysosomes (red) was observed under the lens. As shown in FIG. 2, the co-localization trend was strong at 0.5h, and was reduced at 1h, and hepatic stellate cell targeted exosomes had completely escaped lysosomes at 2 h.
Example 3 hepatic stellate cell targeting exosomes have greater targeting to hepatic stellate cells
The hepatic stellate cells obtained in example 1 were treated by encapsulating fluorescent dye coumarin 6 (C6) in the same manner as the hepatic stellate cells obtained in example 1 and the plasma-derived exosomes of non-tumor patients, and the uptake of the exosomes (green) by Lx2 was observed under a confocal laser microscope at 2,4, and 6 hours, respectively. As shown in fig. 3, compared with plasma-derived exosomes of non-tumor patients, hepatic stellate cell-targeted exosomes were taken up more by Lx2 within the same time, indicating that hepatic stellate cell-targeted exosomes have a stronger targeting to hepatic stellate cells.
Example 4 hepatic stellate cell targeted drug-loaded exosomes have greater inhibition of hepatic stellate cell activation
Hepatic stellate cell Lx2 is treated by hepatic stellate cell targeted exosomes with different concentrations respectively, and the hepatic stellate cell activation marker protein alpha-SMA is detected after 48 hours. Hepatic stellate cell targeting exosomes activates hepatic stellate cells, expressing higher levels of α -SMA, the results are shown in fig. 4.
The hepatic stellate cells Lx2 are treated with free drug Pirfenidone (PF), non-tumor patient plasma source exosome pirfenidone (PF@npE) and pancreatic cancer cell source exosome pirfenidone (PF@PCCE) respectively, cellular proteins are collected after 48 hours, and the alpha-SMA is detected, so that the drug group carrying pirfenidone can reduce the expression level of the alpha-SMA, the PF@PCCE group reduces the expression of the alpha-SMA more, and the hepatic stellate cell targeted drug-carrying exosome is shown to inhibit hepatic stellate cell activation more, and the result is shown in figure 5.
Example 5 hepatic stellate cell targeted drug-loaded exosome safety assessment
The drug-loaded exosomes in example 4 were treated with hepatic stellate cells Lx2 and tumor cells mT7 at different concentrations, respectively, and after 48 hours, CCK8 proliferation assay was performed, and no significant inhibition of proliferation of both cells was found by the drug-loaded exosomes, demonstrating the safety of the drug-loaded exosomes, and the results are shown in fig. 6.
The experimental result shows that the hepatic stellate cell targeted drug-loaded exosome provided by the invention has stronger targeting to hepatic stellate cells, can inhibit hepatic stellate cell activation, achieves the effect of inhibiting the formation of a pre-transfer fibrosis microenvironment, and has the potential of inhibiting pancreatic cancer hepatic metastasis.
The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the invention without departing from the principles thereof are intended to be within the scope of the invention as set forth in the following claims.
Claims (4)
1. A drug-loaded exosome which targets hepatic stellate cells and inhibits activation thereof, wherein the drug-loaded exosome is obtained by introducing an anti-fibrosis drug for inhibiting activation of hepatic stellate cells into an exosome having hepatic stellate cell targeting derived from a pancreatic cancer cell line;
the anti-fibrosis drug for inhibiting hepatic stellate cell activation is pirfenidone.
2. A drug-loaded exosome targeting hepatic stellate cells and inhibiting their activation according to claim 1, wherein the source of the pancreatic cancer cell line is human pancreatic cancer cell line MIA-Paca2 or mouse pancreatic cancer cell line mT7.
3. A drug-loaded exosome targeting hepatic stellate cells and inhibiting their activation according to claim 1, prepared by the method of:
s1, culturing pancreatic cancer cells, collecting a conditional culture medium, and obtaining exosomes derived from the pancreatic cancer cells after differential centrifugation, ultrafiltration concentration, super-separation or exosome extraction reagent incubation;
s2, after the exosome precipitation is resuspended by PBS, a pirfenidone solution is added, and the target product is obtained after circulating freeze thawing, probe ultrasound, water bath balancing and ultrafiltration of superfluous free medicine.
4. Use of a drug-loaded exosome that targets and inhibits activation of hepatic stellate cells as claimed in any one of claims 1 to 3 in the manufacture of a medicament for the treatment of pancreatic cancer liver metastases.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210578625.0A CN115475250B (en) | 2022-05-26 | 2022-05-26 | Drug-loaded exosome targeting hepatic stellate cells and inhibiting activation thereof, and preparation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210578625.0A CN115475250B (en) | 2022-05-26 | 2022-05-26 | Drug-loaded exosome targeting hepatic stellate cells and inhibiting activation thereof, and preparation and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115475250A CN115475250A (en) | 2022-12-16 |
| CN115475250B true CN115475250B (en) | 2024-03-26 |
Family
ID=84420517
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210578625.0A Active CN115475250B (en) | 2022-05-26 | 2022-05-26 | Drug-loaded exosome targeting hepatic stellate cells and inhibiting activation thereof, and preparation and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115475250B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115778953A (en) * | 2022-12-19 | 2023-03-14 | 中国人民解放军海军军医大学第一附属医院 | Application of pirfenidone in medicine for treating pancreatic diseases |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016123556A1 (en) * | 2015-01-30 | 2016-08-04 | The Johns Hopkins University | Extracellular vesicles for agent delivery |
| WO2018093233A1 (en) * | 2016-11-21 | 2018-05-24 | 성균관대학교산학협력단 | Composition containing adipose stem cell-derived exosomes as active ingredient for preventing or treating liver fibrosis |
| CN112469423A (en) * | 2018-05-09 | 2021-03-09 | 儿童医学中心公司 | Mononuclear cell treated by mesenchymal stromal cell exosome and application thereof |
| CN113388571A (en) * | 2021-06-22 | 2021-09-14 | 姜海涛 | Liver targeted drug-loaded exosome and application and drug for treating liver diseases |
| CN113521294A (en) * | 2021-06-22 | 2021-10-22 | 姜海涛 | Stomach targeting drug-loaded exosome and application and drug for treating stomach diseases |
| CN113925856A (en) * | 2021-10-13 | 2022-01-14 | 郑州大学 | Preparation method and application of bionic nano-drug targeting hepatic stellate cells |
| CN114269358A (en) * | 2019-07-31 | 2022-04-01 | 上海圣特佳健康科技发展有限公司 | Therapeutic agents for fibrotic, inflammatory, and/or aging diseases |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11458097B2 (en) * | 2016-03-30 | 2022-10-04 | The University Of North Carolina At Chapel Hill | Biological agent-exosome compositions and uses thereof |
| TWI704923B (en) * | 2017-06-26 | 2020-09-21 | 台灣基督長老教會馬偕醫療財團法人馬偕紀念醫院 | Formulation comprising extracellular vesicles, method for producing the same, and uses thereof |
-
2022
- 2022-05-26 CN CN202210578625.0A patent/CN115475250B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016123556A1 (en) * | 2015-01-30 | 2016-08-04 | The Johns Hopkins University | Extracellular vesicles for agent delivery |
| WO2018093233A1 (en) * | 2016-11-21 | 2018-05-24 | 성균관대학교산학협력단 | Composition containing adipose stem cell-derived exosomes as active ingredient for preventing or treating liver fibrosis |
| CN112469423A (en) * | 2018-05-09 | 2021-03-09 | 儿童医学中心公司 | Mononuclear cell treated by mesenchymal stromal cell exosome and application thereof |
| CN114269358A (en) * | 2019-07-31 | 2022-04-01 | 上海圣特佳健康科技发展有限公司 | Therapeutic agents for fibrotic, inflammatory, and/or aging diseases |
| CN113388571A (en) * | 2021-06-22 | 2021-09-14 | 姜海涛 | Liver targeted drug-loaded exosome and application and drug for treating liver diseases |
| CN113521294A (en) * | 2021-06-22 | 2021-10-22 | 姜海涛 | Stomach targeting drug-loaded exosome and application and drug for treating stomach diseases |
| CN113925856A (en) * | 2021-10-13 | 2022-01-14 | 郑州大学 | Preparation method and application of bionic nano-drug targeting hepatic stellate cells |
Non-Patent Citations (4)
| Title |
|---|
| Hepatic Stellate Cells: Partners in Crime for Liver Metastases?;Ningling Kang等;HEPATOLOGY;707-713 * |
| Pancreatic Premalignant Lesions Secrete Tissue Inhibitor of Metalloproteinases-1, Which Activates Hepatic Stellate Cells Via CD63 Signaling to Create a Premetastatic Niche in the Liver;Barbara Grünwald等;Gastroenterology;第151卷(第5期);1011-1024 * |
| Pirfenidone-loaded exosomes derived from pancreatic ductal adenocarcinoma cells alleviate fibrosis of premetastatic niches to inhibit liver metastasis;Jing Zhao等;Biomater. Sci.;6614–6626 * |
| 胰腺癌来源的外泌体在肝转移中的作用及其机制研究;余泽前;万方;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115475250A (en) | 2022-12-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhou et al. | Pancreatic cancer-targeting exosomes for enhancing immunotherapy and reprogramming tumor microenvironment | |
| Ye et al. | Bioinspired nanoplatelets for chemo-photothermal therapy of breast cancer metastasis inhibition | |
| Lin et al. | Self-assembled tumor-targeting hyaluronic acid nanoparticles for photothermal ablation in orthotopic bladder cancer | |
| Yang et al. | Chlorin e6 and CRISPR-Cas9 dual-loading system with deep penetration for a synergistic tumoral photodynamic-immunotherapy | |
| Yang et al. | Enzyme-triggered self-assembly of gold nanoparticles for enhanced retention effects and photothermal therapy of prostate cancer | |
| Xu et al. | Synergistic inhibition of breast cancer metastasis by silibinin-loaded lipid nanoparticles containing TPGS | |
| Tian et al. | Tumor exosome mimicking nanoparticles for tumor combinatorial chemo-photothermal therapy | |
| Geng et al. | Active-targeting NIR-II phototheranostics in multiple tumor models using platelet-camouflaged nanoprobes | |
| Wang et al. | Oxygen‐deficient molybdenum oxide nanosensitizers for ultrasound‐enhanced cancer metalloimmunotherapy | |
| Huang et al. | Self-driven nanoprodrug platform with enhanced ferroptosis for synergistic photothermal-IDO immunotherapy | |
| Guo et al. | Fucoidan-functionalized activated platelet-hitchhiking micelles simultaneously track tumor cells and remodel the immunosuppressive microenvironment for efficient metastatic cancer treatment | |
| CN109731106B (en) | Preparation method of compound for treating brain glioma | |
| CN115475250B (en) | Drug-loaded exosome targeting hepatic stellate cells and inhibiting activation thereof, and preparation and application thereof | |
| Liu et al. | Development of injectable thermosensitive polypeptide hydrogel as facile radioisotope and radiosensitizer hotspot for synergistic brachytherapy | |
| Lu et al. | Simultaneous inhibition of breast cancer and its liver and lung metastasis by blocking inflammatory feed-forward loops | |
| Wang et al. | Macrophage-derived implantable vaccine prevents postsurgical tumor recurrence | |
| Zhou et al. | Inspired heat shock protein alleviating prodrug enforces immunogenic photodynamic therapy by eliciting pyroptosis | |
| Han et al. | Inhibition of SerpinB9 to enhance granzyme B-based tumor therapy by using a modified biomimetic nanoplatform with a cascade strategy | |
| Wang et al. | MMP-responsive transformation nanomaterials with IAP antagonist to boost immune checkpoint therapy | |
| CN110960688A (en) | Low-toxicity bionic nano system for improving curative effect of pancreatic cancer and preparation method thereof | |
| JP2020536914A (en) | Autophagy inhibitors and their preparation methods and applications | |
| Zhao et al. | Partial ligand shielding nanoparticles improve pancreatic ductal adenocarcinoma treatment via a multifunctional paradigm for tumor stroma reprogramming | |
| Chen et al. | Leveraging tumor cell ferroptosis for colorectal cancer treatment via nanoelicitor-activated tumoricidal immunity | |
| CN107157950A (en) | A kind of albumin nano granular and its production and use | |
| Du et al. | Tumor-associated macrophage membrane-camouflaged pH-responsive polymeric micelles for combined cancer chemotherapy-sensitized immunotherapy |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |