CN115475250B - 一种靶向肝星状细胞并抑制其激活的载药外泌体及其制备和应用 - Google Patents
一种靶向肝星状细胞并抑制其激活的载药外泌体及其制备和应用 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
- activation
- exosome
- 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
- 210000001808 exosome Anatomy 0.000 title claims abstract description 78
- 210000004024 hepatic stellate cell Anatomy 0.000 title claims abstract description 64
- 229940079593 drug Drugs 0.000 title claims abstract description 51
- 239000003814 drug Substances 0.000 title claims abstract description 51
- 230000004913 activation Effects 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims description 4
- 210000004027 cell Anatomy 0.000 claims abstract description 28
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims abstract description 27
- 201000002528 pancreatic cancer Diseases 0.000 claims abstract description 27
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims abstract description 26
- 208000008443 pancreatic carcinoma Diseases 0.000 claims abstract description 26
- 206010027476 Metastases Diseases 0.000 claims abstract description 20
- 230000009401 metastasis Effects 0.000 claims abstract description 19
- 210000004185 liver Anatomy 0.000 claims abstract description 14
- 230000008685 targeting Effects 0.000 claims abstract description 13
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 9
- 230000002300 anti-fibrosis Effects 0.000 claims abstract 2
- ISWRGOKTTBVCFA-UHFFFAOYSA-N pirfenidone Chemical compound C1=C(C)C=CC(=O)N1C1=CC=CC=C1 ISWRGOKTTBVCFA-UHFFFAOYSA-N 0.000 claims description 18
- 229960003073 pirfenidone Drugs 0.000 claims description 17
- 238000000108 ultra-filtration Methods 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- 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
- 230000003510 anti-fibrotic effect Effects 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
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000003636 conditioned culture medium Substances 0.000 claims description 2
- 238000001085 differential centrifugation Methods 0.000 claims description 2
- 238000011067 equilibration Methods 0.000 claims description 2
- 230000008014 freezing Effects 0.000 claims description 2
- 238000007710 freezing Methods 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- 238000010257 thawing Methods 0.000 claims description 2
- 238000005199 ultracentrifugation Methods 0.000 claims description 2
- 208000019423 liver disease Diseases 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 16
- 206010016654 Fibrosis Diseases 0.000 abstract description 9
- 230000004761 fibrosis Effects 0.000 abstract description 9
- 230000020411 cell activation Effects 0.000 abstract description 6
- 230000035755 proliferation Effects 0.000 abstract description 6
- 238000001262 western blot Methods 0.000 abstract description 6
- 230000015572 biosynthetic process Effects 0.000 abstract description 5
- 238000002474 experimental method Methods 0.000 abstract description 4
- 238000000338 in vitro Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract 1
- 230000002452 interceptive effect Effects 0.000 abstract 1
- 238000004393 prognosis Methods 0.000 abstract 1
- 230000002440 hepatic effect Effects 0.000 description 19
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 4
- 210000003712 lysosome Anatomy 0.000 description 4
- 230000001868 lysosomic effect Effects 0.000 description 4
- 208000005443 Circulating Neoplastic Cells Diseases 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 230000003176 fibrotic effect Effects 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
- 230000001394 metastastic effect Effects 0.000 description 3
- 206010061289 metastatic neoplasm Diseases 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
- 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
- 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
- 239000006143 cell culture medium Substances 0.000 description 2
- 230000008045 co-localization Effects 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
- 238000001493 electron microscopy Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 208000036971 interstitial lung disease 2 Diseases 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 230000002132 lysosomal effect Effects 0.000 description 2
- 201000008129 pancreatic ductal adenocarcinoma Diseases 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
- 230000001225 therapeutic effect Effects 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 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
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 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
- 206010061309 Neoplasm progression Diseases 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
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000004663 cell proliferation 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
- 230000012202 endocytosis Effects 0.000 description 1
- 210000001163 endosome Anatomy 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
- 206010017758 gastric cancer Diseases 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000003112 inhibitor Substances 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
- 238000004519 manufacturing process Methods 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
- 239000002244 precipitate Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000004500 stellate cell Anatomy 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
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000005751 tumor progression 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
本发明提供一种靶向肝星状细胞并抑制其激活的载药外泌体，所述载药外泌体由用于抑制肝星状细胞活化的抗纤维化药物导入到具有肝星状细胞靶向性的外泌体中获得，所述具有肝星状细胞靶向性的外泌体来源于胰腺癌细胞系。本发明提供的肝星状细胞靶向载药外泌体对肿瘤和肝星状细胞的体外增殖实验得到了预期安全的治疗效果，对肝星状细胞干预后的Western blot实验亦表明其可以抑制肝星状细胞活化。利用本发明的肝星状细胞靶向载药外泌体干预纤维化转移前生态位的形成，为抑制胰腺癌的肝转移提供可能，亦为其他肿瘤转移的治疗提供了启示。
Description
技术领域
本发明涉及生物医药技术领域，具体涉及一种靶向肝星状细胞并抑制其激活的载药外泌体及其制备和应用。
背景技术
胰腺导管腺癌(PDAC)是公认最具侵袭性的癌症之一，在所有恶性肿瘤中5年生存率最低，仅仅只有约10％，其恶性程度极高与一系列因素有关，最突出的原因可能在于它的高转移潜能。在所有容易发生远处转移的器官中，最常见的远处转移部位是肝脏(76-80％)。肿瘤转移的“种子和土壤”学说中，癌细胞和远处微环境之间的相互作用优化了循环肿瘤细胞定植的可能。原发肿瘤中的癌细胞在扩散之前，提前在次级器官诱导形成有利于其转移侵袭的微环境，即转移前生态位。
原发肿瘤来源的细胞外囊泡(EV，包括外泌体Exosome)和转移器官的局部间质微环境极大程度上决定了转移的发生。外泌体大小约为40～160nm，起源于内泌体，参与体内的多种生理和包括肿瘤转移在内的病理过程。例如，外泌体内EGFR从胃癌细胞到肝星状细胞的转运，通过增强肝脏中的HGF(肝细胞生长因子)信号通路，促进了肝脏特异性的转移。微环境纤维化的发生具有隐蔽性，循环中的肿瘤细胞黏附在这个微环境上可以被激活，从而促进增殖。因此，急需一种靶向肝星状细胞并抑制其激活的载药外泌体。
发明内容
研究证明，来源于胰腺原发肿瘤的外泌体可以被肝星状细胞(HSCs)摄取，上调HSCs中α-SMA和纤维连接蛋白的产生，从而在肝脏形成纤维化的微环境，这揭示了胰腺癌原发肿瘤来源外泌体可以克服微环境纤维化发生的隐蔽性，具有精准靶向到达肝星状细胞的潜力。本发明的目的是提供一种靶向肝星状细胞并抑制其激活的载药外泌体及其制备和应用，以干预主要由肝星状细胞活化引起的纤维化微环境的发生，从而对胰腺癌肝转移起到抑制作用，以解决上述背景中提出的问题。
为实现上述目的，本发明采用以下技术方案：
一种靶向肝星状细胞并抑制其激活的载药外泌体，所述载药外泌体由用于抑制肝星状细胞活化的抗纤维化药物导入到具有肝星状细胞靶向性的外泌体中获得，所述具有肝星状细胞靶向性的外泌体来源于胰腺癌细胞系。
为优化上述技术方案，采取的具体措施还包括：
进一步地，所述胰腺癌细胞系的来源包括但不限于人体胰腺癌细胞系MIA-Paca2、小鼠胰腺癌细胞系mT7。
进一步地，所述用于抑制肝星状细胞活化的抗纤维化药物包括但不限于吡非尼酮。
进一步地，所述靶向肝星状细胞并抑制其激活的载药外泌体通过以下方法制备：
S1、培养胰腺癌细胞，收取条件培养基，经过差速离心、超滤浓缩、超离或加入外泌体提取试剂孵育，离心后获取胰腺癌细胞来源外泌体；
S2、PBS重悬外泌体沉淀后，加入吡非尼酮溶液，经过循环冻融、探头超声、水浴平衡、超滤多余游离药物后获得目的产物。
进一步地，所述靶向肝星状细胞并抑制其激活的载药外泌体用于制备治疗胰腺癌肝转移药物；具体的，所述载药外泌体通过抑制纤维化微环境形成从而抑制肿瘤肝转移，胰腺癌细胞来源外泌体可以主动靶向肝星状细胞，载吡非尼酮后可以对肝星状细胞的活化起到抑制作用。
本发明的有益效果是：本发明提供的肝星状细胞靶向载药外泌体将纤维化药物靶向运输至肝星状细胞；其中，外泌体来源于胰腺癌细胞系，具有良好的相容性、通透性、天然稳定性、低免疫原性和毒性等优点；纤维化药物为吡非尼酮(PF)，PF是一种对特发性肺纤维化(IPF)有治疗作用的药理化合物，它具有调节纤维化生长因子，从而减弱成纤维细胞的增殖、胶原和纤维连接蛋白的合成，以及细胞外基质的沉积的作用；胰腺癌细胞系的外泌体载吡非尼酮的过程经过一系列的破膜、重构后，其促进肿瘤进展的活性成分构成已被改变，生物安全性得到了保障；纤维化药物运输至肝星状细胞后，干预适合循环肿瘤细胞定植的生态位的形成，从而抑制胰腺癌的肝转移。体外增殖实验得到了预期安全的治疗效果，对肝星状细胞干预后的Western blot实验亦表明其可以抑制肝星状细胞活化。利用本发明的肝星状细胞靶向载药外泌体干预纤维化转移前生态位的形成，为抑制胰腺癌的肝转移提供可能，亦为其他肿瘤转移的治疗提供了启示。
附图说明
图1为靶向肝星状细胞并抑制其激活的载药外泌体的粒径检测和透射电镜图片；
图2为靶向肝星状细胞并抑制其激活的载药外泌体在肝星状细胞内溶酶体逃逸结果；
图3为靶向肝星状细胞并抑制其激活的载药外泌体在肝星状细胞内的摄取；
图4为靶向肝星状细胞并抑制其激活的载药外泌体激活后标志蛋白的Westernblot检测；
图5为肝星状细胞活化被抑制的Western blot检测；
图6为靶向肝星状细胞并抑制其激活的载药外泌体在肿瘤细胞、肝星状细胞增殖安全性方面的CCK8检测。
具体实施方式
实施例1外泌体分离收集与载药
采用外泌体提取试剂盒(河南贝贝科技公司)提取收集人胰腺癌细胞分泌的外泌体MIA-Paca-2。
MIA-Paca-2细胞培养液为无外泌体血清和DMEM培养基培养；300g离心力下离心5min，取上清液；2000g离心力下离心20min，取上清液；再在12000g离心力下离心35min，取上清液，弃去细胞碎片及沉淀物；将离心后的细胞培养液用100kD超滤管，2000g离心力下离心15min，取上室液体，过0.22μm滤膜，加入1/3体积外泌体提取试剂，混匀后置于4℃摇床孵育1h，然后在12000rpm离心力下离心20min，沉淀用无菌PBS重悬收集，4℃短期保存，即得肝星状细胞靶向外泌体。BCA蛋白定量法粗测外泌体蛋白浓度为10μg/μL。
获得的外泌体溶液中按体积比1:1加入吡非尼酮溶液(10mg/mL，溶剂PBS)，液氮冻5min，室温平衡10min，重复2次；冰浴下探头超声5min(超声3s，间隔2s，最高温度30℃，功率9％)，室温平衡10min，重复2次；37℃水浴平衡1h恢复膜稳定性；加入100kD超滤管，2000g离心力下离心5min，加入PBS冲洗，2000g离心力下离心5min，取上室液体，即得到肝星状细胞靶向载药外泌体。加入DMSO稀释并探头超声后使用紫外分光光度计在310nm处测量吸光度，根据标准曲线计算出吡非尼酮浓度3-4μg/μL。
对分离收集到的肝星状细胞靶向外泌体和肝星状细胞靶向载药外泌体进行标志蛋白的Western blot、电镜、粒径检测。Western blot结果表明外泌体标志物CD63、TSG101均高表达；电镜结果表明肝星状细胞靶向外泌体和肝星状细胞靶向载药外泌体均可见双凹圆盘状囊泡；粒径分析检测到粒子大小在60-120nm，具体结果如图1所示。
实施例2肝星状细胞靶向外泌体的溶酶体逃逸过程
纳米级别药物进入细胞内往往采取胞吞形式，并被溶酶体摄取，从溶酶体中释放出来后可在胞浆中发挥作用。将实施例1中获得的肝星状细胞靶向外泌体用同样方式包载荧光染料香豆素6(C6)，加入肝星状细胞Lx2中进行处理，分别在0.5，1，2h加入溶酶体示踪试剂，镜下观察肝星状细胞靶向外泌体(绿色)与溶酶体(红色)的位置关系。结果如图2所示，在0.5h的时候，二者共定位趋势较强，在1h的时候，共定位趋势即减弱，在2h的时候，肝星状细胞靶向外泌体已完全逃逸出溶酶体。
实施例3肝星状细胞靶向外泌体对肝星状细胞具有更强靶向性
将实施例1中获得的肝星状细胞靶向外泌体、非肿瘤病人血浆来源外泌体用同样方式包载荧光染料香豆素6(C6)，加入肝星状细胞Lx2中进行处理，分别在2，4，6h的时候，在激光共聚焦显微镜下观察Lx2摄取外泌体(绿色)的情况。如图3所示，相较于非肿瘤病人血浆来源外泌体，在相同时间内，肝星状细胞靶向外泌体被Lx2摄取更多，表明肝星状细胞靶向外泌体对肝星状细胞具有更强的靶向性。
实施例4肝星状细胞靶向载药外泌体对肝星状细胞活化具有更强抑制作用
分别用不同浓度的肝星状细胞靶向外泌体对肝星状细胞Lx2进行处理，48h后对肝星状细胞活化的标志蛋白α-SMA进行检测。肝星状细胞靶向外泌体可激活肝星状细胞，表达更高水平α-SMA，结果如图4所示。
分别用游离药物吡非尼酮(PF)、非肿瘤病人血浆来源外泌体载吡非尼酮(PF@npE)、胰腺癌细胞来源外泌体载吡非尼酮(PF@PCCE)处理肝星状细胞Lx2，48h后收集细胞蛋白，对α-SMA进行检测，发现载吡非尼酮药物组可降低α-SMA的表达水平，且PF@PCCE组降低α-SMA表达更多，表现出肝星状细胞靶向载药外泌体更强的抑制肝星状细胞活化作用，结果如图5所示。
实施例5肝星状细胞靶向载药外泌体安全性评估
将实施例4中的各载药外泌体分别以不同浓度处理肝星状细胞Lx2和肿瘤细胞mT7，48h后进行CCK8增殖检测，发现载药外泌体对两种细胞的增殖没有显著抑制作用，证明了载药外泌体的安全性，结果如图6所示。
上述实验结果表明，本发明提供的肝星状细胞靶向载药外泌体对肝星状细胞具有更强的靶向性，同时可抑制肝星状细胞活化，达到抑制转移前纤维化微环境形成的作用，具有抑制胰腺癌肝转移的潜能。
以上仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，应视为本发明的保护范围。
Claims (4)
1.一种靶向肝星状细胞并抑制其激活的载药外泌体，其特征在于，所述载药外泌体由用于抑制肝星状细胞活化的抗纤维化药物导入到具有肝星状细胞靶向性的外泌体中获得，所述具有肝星状细胞靶向性的外泌体来源于胰腺癌细胞系；
所述用于抑制肝星状细胞活化的抗纤维化药物为吡非尼酮。
2.根据权利要求1所述的一种靶向肝星状细胞并抑制其激活的载药外泌体，其特征在于，所述胰腺癌细胞系的来源为人体胰腺癌细胞系MIA-Paca2或小鼠胰腺癌细胞系mT7。
3.根据权利要求1所述的一种靶向肝星状细胞并抑制其激活的载药外泌体，其特征在于，通过以下方法制备：
S1、培养胰腺癌细胞，收取条件培养基，经过差速离心、超滤浓缩、超离或加入外泌体提取试剂孵育，离心后获取胰腺癌细胞来源外泌体；
S2、PBS重悬外泌体沉淀后，加入吡非尼酮溶液，经过循环冻融、探头超声、水浴平衡、超滤多余游离药物后获得目的产物。
4.如权利要求1-3任一项所述的靶向肝星状细胞并抑制其激活的载药外泌体在制备治疗胰腺癌肝转移药物中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210578625.0A CN115475250B (zh) | 2022-05-26 | 2022-05-26 | 一种靶向肝星状细胞并抑制其激活的载药外泌体及其制备和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210578625.0A CN115475250B (zh) | 2022-05-26 | 2022-05-26 | 一种靶向肝星状细胞并抑制其激活的载药外泌体及其制备和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115475250A CN115475250A (zh) | 2022-12-16 |
| CN115475250B true CN115475250B (zh) | 2024-03-26 |
Family
ID=84420517
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210578625.0A Active CN115475250B (zh) | 2022-05-26 | 2022-05-26 | 一种靶向肝星状细胞并抑制其激活的载药外泌体及其制备和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115475250B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115778953A (zh) * | 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 | 吡非尼酮在治疗胰腺疾病的药物中的应用 |
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 (ko) * | 2016-11-21 | 2018-05-24 | 성균관대학교산학협력단 | 지방줄기세포유래 엑소좀을 유효성분으로 포함하는 간 섬유증 예방 또는 치료용 조성물 |
| CN112469423A (zh) * | 2018-05-09 | 2021-03-09 | 儿童医学中心公司 | 经间充质基质细胞外排体处理的单核细胞及其用途 |
| CN113388571A (zh) * | 2021-06-22 | 2021-09-14 | 姜海涛 | 一种肝脏靶向载药外泌体及应用和治疗肝部疾病药物 |
| CN113521294A (zh) * | 2021-06-22 | 2021-10-22 | 姜海涛 | 一种胃靶向载药外泌体及应用和治疗胃部疾病药物 |
| CN113925856A (zh) * | 2021-10-13 | 2022-01-14 | 郑州大学 | 一种靶向肝星状细胞的仿生纳米药物的制备方法及其应用 |
| CN114269358A (zh) * | 2019-07-31 | 2022-04-01 | 上海圣特佳健康科技发展有限公司 | 针对纤维化、炎症、和/或老化疾病的治疗药物 |
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 (zh) * | 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 | 包含胞外囊泡之製劑、用以製備該製劑之方法及其用途 |
-
2022
- 2022-05-26 CN CN202210578625.0A patent/CN115475250B/zh 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 (ko) * | 2016-11-21 | 2018-05-24 | 성균관대학교산학협력단 | 지방줄기세포유래 엑소좀을 유효성분으로 포함하는 간 섬유증 예방 또는 치료용 조성물 |
| CN112469423A (zh) * | 2018-05-09 | 2021-03-09 | 儿童医学中心公司 | 经间充质基质细胞外排体处理的单核细胞及其用途 |
| CN114269358A (zh) * | 2019-07-31 | 2022-04-01 | 上海圣特佳健康科技发展有限公司 | 针对纤维化、炎症、和/或老化疾病的治疗药物 |
| CN113388571A (zh) * | 2021-06-22 | 2021-09-14 | 姜海涛 | 一种肝脏靶向载药外泌体及应用和治疗肝部疾病药物 |
| CN113521294A (zh) * | 2021-06-22 | 2021-10-22 | 姜海涛 | 一种胃靶向载药外泌体及应用和治疗胃部疾病药物 |
| CN113925856A (zh) * | 2021-10-13 | 2022-01-14 | 郑州大学 | 一种靶向肝星状细胞的仿生纳米药物的制备方法及其应用 |
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 (zh) | 2022-12-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | An exosome-like programmable-bioactivating paclitaxel prodrug nanoplatform for enhanced breast cancer metastasis inhibition | |
| Zhou et al. | Pancreatic cancer-targeting exosomes for enhancing immunotherapy and reprogramming tumor microenvironment | |
| Cao et al. | Activating cGAS-STING pathway with ROS-responsive nanoparticles delivering a hybrid prodrug for enhanced chemo-immunotherapy | |
| Lin et al. | Self-assembled tumor-targeting hyaluronic acid nanoparticles for photothermal ablation in orthotopic bladder cancer | |
| Luan et al. | Acid‐responsive aggregated gold nanoparticles for radiosensitization and synergistic chemoradiotherapy in the treatment of esophageal cancer | |
| Liu et al. | A potential target associated with both cancer and cancer stem cells: a combination therapy for eradication of breast cancer using vinorelbine stealthy liposomes plus parthenolide stealthy liposomes | |
| Guan et al. | Photo-triggered gadofullerene: enhanced cancer therapy by combining tumor vascular disruption and stimulation of anti-tumor immune responses | |
| Hu et al. | Alantolactone induces concurrent apoptosis and GSDME-dependent pyroptosis of anaplastic thyroid cancer through ROS mitochondria-dependent caspase pathway | |
| Zhang et al. | Biodegradable Hypocrellin B nanoparticles coated with neutrophil membranes for hepatocellular carcinoma photodynamics therapy effectively via JUNB/ROS signaling | |
| Hu et al. | 9-amino acid cyclic peptide-decorated sorafenib polymeric nanoparticles for the efficient in vitro nursing care analysis of hepatocellular carcinoma | |
| Han et al. | Inhibition of SerpinB9 to enhance granzyme B-based tumor therapy by using a modified biomimetic nanoplatform with a cascade strategy | |
| Cao et al. | Enzyme-induced morphological transformation of self-assembled peptide nanovehicles potentiates intratumoral aggregation and inhibits tumour immunosuppression | |
| CN115475250B (zh) | 一种靶向肝星状细胞并抑制其激活的载药外泌体及其制备和应用 | |
| Xu et al. | A biomimetic nanodrug self-assembled from small molecules for enhanced ferroptosis therapy | |
| Yu et al. | Coordination-driven FBXW7 DNAzyme-Fe nanoassembly enables a binary switch of breast cancer cell cycle checkpoint responses for enhanced ferroptosis-radiotherapy | |
| Wang et al. | Engineered liposomes targeting hepatic stellate cells overcome pathological barriers and reverse liver fibrosis | |
| Yu et al. | Chlorin e6-induced photodynamic effect facilitates immunogenic cell death of lung cancer as a result of oxidative endoplasmic reticulum stress and DNA damage | |
| Cao et al. | Lipid core-shell nanoparticles co-deliver FOLFOX regimen and siPD-L1 for synergistic targeted cancer treatment | |
| Peng et al. | Biomineralized nanomedicine for enhanced cancer therapy through in situ toxification and amplified Ca2+ overload | |
| Yao et al. | Iridium nanozyme-mediated photoacoustic imaging-guided NIR-II photothermal therapy and tumor microenvironment regulation for targeted eradication of cancer stem cells | |
| Qi et al. | Engineering multifunctional thylakoid as an oxygen self-supplying photosensitizer for esophageal squamous cell carcinoma-targeted photodynamic therapy | |
| Sha et al. | Mn-CaCO3-based nanosystem for augmented sonodynamic-chemodynamic immunotherapy via PI3K/Akt signaling pathway | |
| Kong et al. | Ultrasmall Polyphenol‐NAD+ Nanoparticle‐Mediated Renal Delivery for Mitochondrial Repair and Anti‐Inflammatory Treatment of AKI‐to‐CKD Progression | |
| Yin et al. | Bio-responsive Au-miR-183 inhibitor enhances immunotherapy in hepatocellular carcinoma by inducing immunogenic cell death | |
| Guo et al. | Tea polyphenol-engineered hybrid cellular nanovesicles for cancer immunotherapy and androgen deprivation therapy |
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 |