CN114949244A - 一种靶向改造的红细胞外泌体 - Google Patents
一种靶向改造的红细胞外泌体 Download PDFInfo
- Publication number
- CN114949244A CN114949244A CN202210519844.1A CN202210519844A CN114949244A CN 114949244 A CN114949244 A CN 114949244A CN 202210519844 A CN202210519844 A CN 202210519844A CN 114949244 A CN114949244 A CN 114949244A
- Authority
- CN
- China
- Prior art keywords
- exosome
- erythrocyte
- cap
- mir
- targeting
- 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
- 210000001808 exosome Anatomy 0.000 title claims abstract description 86
- 210000003743 erythrocyte Anatomy 0.000 title claims abstract description 53
- 230000008685 targeting Effects 0.000 title claims abstract description 29
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 35
- 108091060382 miR-140 stem-loop Proteins 0.000 claims abstract description 28
- LVNGJLRDBYCPGB-UHFFFAOYSA-N 1,2-distearoylphosphatidylethanolamine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(COP([O-])(=O)OCC[NH3+])OC(=O)CCCCCCCCCCCCCCCCC LVNGJLRDBYCPGB-UHFFFAOYSA-N 0.000 claims description 16
- 201000008482 osteoarthritis Diseases 0.000 claims description 14
- 102000007079 Peptide Fragments Human genes 0.000 claims description 8
- 108010033276 Peptide Fragments Proteins 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 7
- 239000002131 composite material Substances 0.000 claims description 6
- 229920001184 polypeptide Polymers 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 4
- 238000012546 transfer Methods 0.000 claims description 4
- 230000000694 effects Effects 0.000 abstract description 9
- 210000004027 cell Anatomy 0.000 description 14
- 241000700159 Rattus Species 0.000 description 12
- 210000001612 chondrocyte Anatomy 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 9
- 239000008055 phosphate buffer solution Substances 0.000 description 8
- 210000000845 cartilage Anatomy 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 6
- 239000007924 injection Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 210000000629 knee joint Anatomy 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 210000004417 patella Anatomy 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 210000000281 joint capsule Anatomy 0.000 description 3
- 210000003041 ligament Anatomy 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 210000002487 multivesicular body Anatomy 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- YODBPLSWNJMZOJ-BPUTZDHNSA-N Asp-Trp-Arg Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CC(=O)O)N YODBPLSWNJMZOJ-BPUTZDHNSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- JLMZKEQFMVORMA-SRVKXCTJSA-N Pro-Pro-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 JLMZKEQFMVORMA-SRVKXCTJSA-N 0.000 description 2
- POQFNPILEQEODH-FXQIFTODSA-N Pro-Ser-Ala Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O POQFNPILEQEODH-FXQIFTODSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- VHRLUTIMTDOVCG-PEDHHIEDSA-N Val-Ile-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)NC(=O)[C@H](C(C)C)N VHRLUTIMTDOVCG-PEDHHIEDSA-N 0.000 description 2
- 108010060035 arginylproline Proteins 0.000 description 2
- 108010036533 arginylvaline Proteins 0.000 description 2
- 206010003246 arthritis Diseases 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 108010031424 isoleucyl-prolyl-proline Proteins 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 108091070501 miRNA Proteins 0.000 description 2
- 239000002679 microRNA Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 238000000035 BCA protein assay Methods 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102100027995 Collagenase 3 Human genes 0.000 description 1
- 108050005238 Collagenase 3 Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 1
- 102000055008 Matrilin Proteins Human genes 0.000 description 1
- 108010072582 Matrilin Proteins Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108020005196 Mitochondrial DNA Proteins 0.000 description 1
- 108091093105 Nuclear DNA Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 210000003195 fascia Anatomy 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- -1 salt ions Chemical class 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- 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
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- 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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Physical Education & Sports Medicine (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Rheumatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明涉及一种靶向改造的红细胞外泌体,其具有靶向递送miR‑140的效果,具体地,采用靶向肽段对红细胞外泌体进行化学修饰。
Description
技术领域
本发明涉及一种靶向改造的红细胞外泌体,其具有靶向递送miR-140的效果。
背景技术
外泌体(EVs)是内涵体来源的,经由多泡体与细胞膜融合,从而释放到细胞外的细胞外囊泡。多泡体与质膜融合后,其中的腔内小体释放到细胞外即为外泌体。外泌体携带多种蛋白质、脂质和核酸等生物活性物质,其直径为50-150nm。研究表明,外泌体是一种极具应用价值的药物递送载体。特别是红细胞来源外泌体是非常有临床转化潜力的外泌体来源。红细胞是人体内最丰富的细胞类型,并且红细胞在多年的常规输血应用已被证明是安全的,无核的红细胞缺乏DNA从而不会引起外源基因转移潜在风险。
研究表明,miR-140具有抑制骨关节炎症状的特异性小分子RNA,靶向递送miR-140可以抑制MMP-13对软骨基质中二型胶原的降解,从而抑制软骨细胞的病变,起到治疗骨关节炎的作用。
CN109966506A公开了一种基因工程改造外泌体递送miRNA-140靶向治疗骨性关节炎的方法。然而,基因工程改造有核细胞来源的细胞外泌体存在基因水平转移的风险,且无法直接使用人源的外泌体。
CN111821416A公开了一种Gp350蛋白修饰的荷载化疗药物的红细胞外泌体在血液肿瘤治疗中的应用,是从EBV病毒基因组扩增出Gp350基因,需要特殊的仪器设备和实验条件,操作流程复杂。
发明内容
为了解决上述问题,本发明提供了一种靶向肽段及经所述靶向肽段改造的红细胞外泌体,以及所述靶向改造红细胞外泌体在制备治疗疾病药物中的应用,所述经靶向肽段改造的红细胞外泌体可以高效加载miR-140并靶向递送miRNA-140至治疗部位。
本发明采用靶向肽段对红细胞外泌体进行化学修饰,获得一种经改造的红细胞外泌体,所述靶向肽段为二硬脂酰磷脂酰乙醇胺(DSPE)和亲和肽(CAP)的复合肽段,本发明的靶向肽段具有脂质的二硬脂酰磷脂酰乙醇胺(DSPE)部分,其能够***外泌体的脂双层膜结构中,从而将亲和肽(CAP)固定到外泌体膜表面,使其能够高效的与miR-140结合。
本发明提供一种靶向肽段,所述靶向肽段为二硬脂酰磷脂酰乙醇胺(DSPE)和亲和肽(CAP)的复合肽段。
优选的,所述亲和肽(CAP)的氨基酸序列为:DWRVIIPPRPSA。
优选的,所述二硬脂酰磷脂酰乙醇胺(DSPE)和亲和肽(CAP)的复合肽段为DSPE-PEG2000-CAP多肽。
本发明提供一种经改造的红细胞外泌体,其特征在于,所述改造为采用靶向肽段对红细胞外泌体进行化学修饰。
优选的,所述靶向肽段为二硬脂酰磷脂酰乙醇胺(DSPE)和亲和肽(CAP)的复合肽段。
优选的,所述亲和肽(CAP)的氨基酸序列为:DWRVIIPPRPSA。
更优选的,所述二硬脂酰磷脂酰乙醇胺(DSPE)和亲和肽(CAP)的复合肽段为DSPE-PEG2000-CAP多肽。
本发明同时提供一种加载miR-140的外泌体,其特在于,所述加载miR-140的外泌体由上述经改造的红细胞外泌体与miR-140结合而成。
优选的,通过电穿孔仪把miR-140电转到经改造的红细胞外泌体完成所述结合。
本发明同时提供所述经改造的红细胞外泌体以及加载miR-140的外泌体在制备治疗骨关节炎药物中的应用。
与现有技术相比,本发明的有益效果是:本发明的靶向肽段可以与外泌体有效固定并高效结合miRNA,经过靶向修饰的红细胞外泌体可提高体外miRNA递送至软骨细胞的效率;外周血红细胞是体内最丰富且易获得的细胞类型,而且红细胞缺乏核DNA及线粒体DNA,不会引起受试者基因转移的风险;此外,异体输血具有很高的安全性,红细胞来源的胞外泌体是红细胞的天然成分,因此可打破自体限制,具有高度的生物兼容性。
附图说明
图1为化学改造方法构建新型的红细胞外泌体递送体系流程图;
图2为红细胞外泌体靶向递送体系的鉴定及表征图:(A)Western blot检测红细胞外泌体改造后相关蛋白表达情况(B)纳米粒径分析仪检测红细胞外泌体改造前后粒径。(C)冷冻电镜鉴定红细胞外泌体改造前后形态;
图3为红细胞外泌体靶向递送miR-140至软骨细胞效率图;
图4为红细胞外泌体靶向递送miR-140至大鼠关节腔后,外泌体在关节的软骨组织层分布图。
图5和图6为红细胞外泌体靶向递送miR-140至大鼠骨关节腔后,在关节的软骨细胞中的效果图。
图7为红细胞外泌体靶向递送miR-140至关节炎模型大鼠关节腔中,关节的H&E染色结果图。
具体实施方式
为了加深对本发明的理解,下面将结合实施例对本发明做进一步详细描述,该实施例仅用于解释本发明,并不对保护范围构成限定。
所有人血样本均经过江苏省中医院(中国南京)批准获得,所有样本均按照该医院的道德准则使用,所有受检者在采集样本前均表示知情同意。
所用的仪器设备、耗材和试剂除特别说明以外,均为市售商品化产品。
实施例1
红细胞的分离及红细胞外泌体提取,获得RBC-Exo
抽取人外周血10ml置入抗凝血离心管中,沉淀后离心,1500r/min离心5min,去掉上层血浆。然后加入等量的生理盐水(0.9%NaCl),混匀,1500r/min离心5min,去上清。再用无D-Hank's液(不含Ca2+、Mg2+、酚红)溶液将红细胞洗涤3次,每次1500r/min离心5min。再用白细胞过滤器(PALL,USA)过滤残余的白细胞后,获得红细胞。
用无Ca2+、Mg2+Hank’s溶液稀释,红细胞置入培养皿中与Ca2+载体(10mM,Abcam,England)按1000:1的比例共孵育37℃培养16个小时。收集红细胞培养上清,依次按照600g离心10min、2000g离心20min去除细胞和凋亡碎片、10000g离心30min去除更大的囊泡,移取上清经0.22μm过滤器过滤后转移到新的离心管中,120000g持续离心70分钟,去掉上清,留下的沉淀用磷酸盐缓冲液(PBS)重悬后,再次以120000g离心70分钟,收集沉淀置于-80℃冰箱备用。
实施例2:红细胞外泌体的软骨靶向性改造,获得CAP-RBC-Exo。
使用DSPE-PEG-Mal(DSPE-PEG 2000马来酰亚胺,购自Merk公司)和合成纯度大于95%的CAP-Cys肽(DWRVIIPPRPSAC,南京肽业公司合成)。将DSPE-PEG-Mal和过量的CAP-Cys,在pH=6.8-7.4条件下过夜反应。根据分子量选择合适透析袋(规格8,000-10,0000道尔顿),将未反应的DSPE-PEG2000-Mal、CAP-Cys以及盐离子去除,后浓缩体积冻干,得到终产物DSPE-PEG2000-CAP多肽(CAP的氨基酸序列为:DWRVIIPPRPSA)备用。1mg纯化的外泌体类似物与DSPE-PEG2000-CAP多肽(100μg/mL)在37℃下孵育1小时,之后通过微量超滤去掉未结合到外泌体膜上的DSPE-PEG2000-CAP多肽。截留在超滤管上面的为DSPE-PEG2000-CAP多肽修饰的软骨靶向外泌体。
实施例3:制备加载miR-140的外泌体
通过电穿孔仪把miR-140(序列为:CAGUGGUUUUACCCUAUGGUAG)电转到第二步所获得的靶向外泌体中,使得外泌体包裹上miR-140,运载miR-140,获得改造后的外泌体:CAP-RBC-Exo-miR140。
可以通过注射包含本实施例制得的CAP-RBC-Exo-miR140靶向外泌体药物到骨关节腔中,从而实现miR-140靶向递送到关节软骨细胞里。
实施例1-3的流程图如图1所示。
实施例4
(A)Western blot检测红细胞外泌体改造后相关蛋白表达情况
将红细胞或红细胞的外泌体加入100ul含有蛋白酶和磷酸酶抑制剂(罗氏公司)的细胞裂解液(RIPA)(Sigma-Aldrich公司)在冰上溶解10分钟,短暂涡旋震荡后,在4℃条件下以10,000g离心清除细胞杂质。使用Pierce BCA蛋白质测定试剂盒(Thermo Scientific)测定来自细胞及外泌体提取的蛋白质浓度。取红细胞及其外泌体的蛋白裂解物(20μg),加入含有还原剂的样品缓冲液中加热10min后上样跑SDS-PAGE胶,利用10%的Bis-Tris凝胶(Bio-Rad)中分离。随后将蛋白胶电转移到0.45-μmPVDF(Bio-Rad)膜上。在三缓冲盐水(TBS)中用20%Odyssey封闭缓冲液(LI-COR)封闭膜,然后在4℃下在10%Odyssey封闭缓冲液中稀释的一抗中孵育过夜。使用的一抗如下:Anti-CD63(ab134045,Abcam)、Anti-CD9(ab92726,Abcam)、Anti-GAPDH antibody(ab9485,Abcam)。之后,在含有0.1%Tween 20(TBST)的TBS中洗涤膜三次,每次10min,然后在HRP山羊抗兔的二抗、在室温(RT)下在TBST中的10%Odyssey封闭缓冲液中稀释1小时。使用LI-COR Odyssey扫描仪和LI-COR Odyssey软件拍照反应条带。实验结果如图2A所示。
(B)纳米粒径分析仪检测红细胞外泌体改造前后粒径。
外泌体颗粒重新悬浮在500μl灭菌的PBS中。使用NanoSight LM10仪器及配套的NTA v3.1软件(Malvern,UK)的测量和量化样品。在测量之前,将外泌体悬浮液超声处理5min,并高速涡旋10秒,以打散聚集体。每帧平均测量17.4±10个粒子。数据是从20°至22℃记录的标准测量中收集的,粘度设置为水(0.940至0.948厘泊),相机级别为16,检测阈值为4,所有其他参数设置为默认值。每个标准测量包括五个持续时间为1分钟(总共5分钟)的视频,并且在每个视频之前注入新鲜样品进行测量。每个时间点使用来自五个视频的有效轨道的流体动力学尺寸分布总和。实验结果如图2B所示。
(C)冷冻电镜鉴定红细胞外泌体改造前后形态。
取5μl外泌体滴加到多孔碳网格(Quantifoil Cu R1.2/1.3),然后将多孔碳网格用Whatman 55mm滤纸吸干1.5秒,并在FEI Vitrobot Mark IV中以液氮温度冷却的液态乙烷浆液中闪蒸冷冻。将多孔碳网格转移到在300kV加速电压下运行并配备Gatan K2 Summit直接电子计数相机的FEI Titan Krios电子显微镜上。通过半自动低剂量采集程序UCSF-Image4以22500倍的标称放大倍数采集超分辨率模式的显微照片。每张图像的总曝光时间为8秒,分为32个子帧。样品上的总累积剂量约为每50个电子。使用IMAGIC-4D和RELION1.3处理图像。实验结果如图2C所示。
实施例5
通过体外实验验证所获得的软骨靶向的外泌体靶向效应
体外细胞实验采用Cy3标记miR-140加载的外泌体,比较CAP靶向的外泌体与非靶向外泌体对软骨细胞的亲和效应,通过Confocal荧光比较软骨细胞细胞摄取荧光标记的外泌体的量。具体步骤如下:
从红细胞分离获得的外泌体颗粒通过电转负载Cy3标记miR-140,然后使用SW41转子以110,000x g离心90分钟去掉未加载到外泌体的游离Cy3标记miR-140。用PBS中重悬外泌体沉淀获得。用PBS洗涤细胞,并在2%多聚甲醛中固定,然后通过显微镜进行进一步分析Cy3-miR-140标记外泌体。
将细胞用胰蛋白酶消化并重新悬浮在1mL无血清培养基中,种到35mmconfocal皿上。待细胞贴壁24小时后向细胞中加入5μL Cy3-miR-140标记外泌体(1mg/ml),然后在37℃、5%CO2下孵育2小时。弃去上清液。用PBS洗涤细胞3次,再4%多聚甲醛固定15分钟,然后在室温下用DAPI染色5分钟。洗涤后,用荧光显微镜(Leica DMI6000B,Solms,Germany)分析细胞。实验结果见图3。
实施例6
通过体内实验验证所获得的软骨靶向的外泌体靶向效应。
该实验验证实施例3制备的CAP-RBC-Exo-miR140是否可以在体内将纳米囊泡递送到软骨细胞内。体内实验采用DiR标记的CAP-RBC-Exo-miR140,DiR是一种荧光染料,可突出外泌体的脂质膜。通过关节内注射,将DiR标记的CAP-RBC-Exo-miR140注射到大鼠关节,并在给药后48小时通过荧光显微镜进行监测。DiR的荧光信号表明,在i.a.注射后,没有CAP的RBC-Exo显示出更广泛的分布并扩散到身体的其他部位。48小时后检查主要器官RBC-Exo-miR140除了在关节腔中,在肾脏和肝脏中也有富集(见图4A)。而CAP-RBC-Exo-miR140都保留在关节腔中(见图4B)。因此,软骨细胞靶向性限制了关节腔内注射后标记的外泌体,而不会明显扩散到其他器官,显示了在临床试验中靶向递送的效果。
接下来,检测实施例5制备的CAP-RBC-Exo-Cy3-miR140是否可以将Cy3-miR-140递送至体内软骨区域内深层嵌入的软骨细胞。通过关节腔注射CAP-RBC-Exo-Cy3-miR140和RBC-Exo-Cy3-miR140制剂至OA大鼠骨关节腔内。大鼠在24或48小时后被解剖,软骨组织被收集、切片、染色和成像。在大鼠骨关节的软骨细胞中观察到的Cy3信号显著高于没有CAP肽的对照外泌体(见图5、图6)。
实施例7
CAP-RBC-Exo-miR140动物实验效果评价。
将实施例3获得的CAP-RBC-Exo-miR140进行关节炎动物模型的骨关节腔注射。本实验选用6周龄SD大鼠,采用内侧半月板不稳定(DMM)手术造模构建膝关节OA模型。由于DMM的手术模型已成为研究创伤后骨关节炎(OA)发病和进展的金标准,我们采用了该模型并开发了OA大鼠。
DMM手术方法:
无菌条件下,膝关节内侧入路,髌骨与胫骨平台内侧2mm做5mm纵行切口,钝性分离皮下肌肉及软组织,眼科剪剪开关节囊。钝性分离髌下脂肪垫。髌骨脱位后,肉眼可直视内侧半月板胫骨韧带,显微手术刀离断内侧半月板胫骨韧带,生理盐水冲洗关节腔,复位髌骨,对损伤韧带进行缝合修补,后关闭关节囊,逐层缝合关节囊,肌肉,筋膜,皮肤。
OA治疗效果检测:组织切片检测软骨缺损情况。DMM手术两周后,通过组织切片检测大鼠软骨缺损情况;连续膝关节腔注射PBS、RBC-Exo-miR140、CAP-RBC-Exo-miR140分别经膝关节注射入骨关节炎模型大鼠关节腔,药物注射四周后,二氧化碳安乐处死大鼠,取双侧膝关节,4%多聚甲醛固定48小时后,样品置于EDTA碱性溶液中脱钙两个月。样品经脱水,包埋,切片后行H&E染色。结果提示和PBS、RBC-Exo-miR140相比,CAP-RBC-Exo-miR140可以更好地治疗OA(见图7)。
显然,上述实施例仅仅是为清楚地说明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
序列表
<110> 南京逸微健华生物科技有限公司
<120> 一种靶向改造的红细胞外泌体
<130> js2022101
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> 人工序列()
<400> 1
Asp Trp Arg Val Ile Ile Pro Pro Arg Pro Ser Ala
1 5 10
<210> 2
<211> 13
<212> PRT
<213> 人工序列()
<400> 2
Asp Trp Arg Val Ile Ile Pro Pro Arg Pro Ser Ala Cys
1 5 10
<210> 3
<211> 22
<212> RNA
<213> 人工序列()
<400> 3
cagugguuuu acccuauggu ag 22
Claims (8)
1.一种靶向肽段,其特征在于,其为二硬脂酰磷脂酰乙醇胺(DSPE)和亲和肽(CAP)的复合肽段。
2.如权利要求1所述的靶向肽段,其特征在于,所述亲和肽(CAP)的氨基酸序列为:DWRVIIPPRPSA。
3.如权利要求1或2所述的靶向肽段,其特征在于,所述二硬脂酰磷脂酰乙醇胺(DSPE)和亲和肽(CAP)的复合肽段为DSPE-PEG2000-CAP多肽。
4.一种经改造的红细胞外泌体,其特征在于,所述改造为采用如权利要求1-3任一项所述的靶向肽段对红细胞外泌体进行化学修饰。
5.一种加载miR-140的外泌体,其特在于,所述加载miR-140的外泌体由权利要求4所述的经改造的红细胞外泌体与miR-140结合而成。
6.如权利要求5所述的外泌体,其特征在于,通过电穿孔仪把miR-140电转到所述经改造的红细胞外泌体完成所述结合。
7.权利要求4所述的经改造的红细胞外泌体在制备治疗骨关节炎药物中的应用。
8.权利要求5所述的加载miR-140的外泌体在制备治疗骨关节炎药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210519844.1A CN114949244A (zh) | 2022-05-12 | 2022-05-12 | 一种靶向改造的红细胞外泌体 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210519844.1A CN114949244A (zh) | 2022-05-12 | 2022-05-12 | 一种靶向改造的红细胞外泌体 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN114949244A true CN114949244A (zh) | 2022-08-30 |
Family
ID=82983493
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210519844.1A Pending CN114949244A (zh) | 2022-05-12 | 2022-05-12 | 一种靶向改造的红细胞外泌体 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114949244A (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109966506A (zh) * | 2019-03-01 | 2019-07-05 | 深圳市第二人民医院 | 基因工程改造外泌体递送miRNA-140靶向治疗骨性关节炎的方法 |
CN110652492A (zh) * | 2019-09-18 | 2020-01-07 | 浙江大学 | 一种载药外泌体及其应用、肝脏疾病药物 |
CN113274509A (zh) * | 2021-05-28 | 2021-08-20 | 广东药科大学 | 一种多肽药物纳米靶向给药***HTPP-Exo-M1-8及其制备方法和应用 |
CN114457038A (zh) * | 2021-11-08 | 2022-05-10 | 仲飙 | 一种载基因外泌体及其制备方法、应用 |
-
2022
- 2022-05-12 CN CN202210519844.1A patent/CN114949244A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109966506A (zh) * | 2019-03-01 | 2019-07-05 | 深圳市第二人民医院 | 基因工程改造外泌体递送miRNA-140靶向治疗骨性关节炎的方法 |
CN110652492A (zh) * | 2019-09-18 | 2020-01-07 | 浙江大学 | 一种载药外泌体及其应用、肝脏疾病药物 |
CN113274509A (zh) * | 2021-05-28 | 2021-08-20 | 广东药科大学 | 一种多肽药物纳米靶向给药***HTPP-Exo-M1-8及其制备方法和应用 |
CN114457038A (zh) * | 2021-11-08 | 2022-05-10 | 仲飙 | 一种载基因外泌体及其制备方法、应用 |
Non-Patent Citations (3)
Title |
---|
丁昆山: "基于巯基-马来酰亚胺迈克尔加成反应的数字高分子的构建", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》, no. 04 * |
方未晶: "髓核靶向性纳米给药***的构建及在椎间盘退变中的应用", 《中国博士学位论文全文数据库工程科技Ⅰ辑》, no. 01 * |
耿旭等: "马来酰亚胺聚谷氨酸天冬氨酸聚合物偶联性能研究", 《河南师范大学学报(自然科学版)》, vol. 41, no. 3, pages 117 - 119 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Borrelli et al. | Extracellular vesicle therapeutics for liver disease | |
Feng et al. | Reversing the surface charge of MSC‐derived small extracellular vesicles by εPL‐PEG‐DSPE for enhanced osteoarthritis treatment | |
US6139836A (en) | Method of encapsulating biologically active agents within erythrocytes, and apparatus therefor | |
KR101720851B1 (ko) | 세포의 지질막에서 유래된 나노소포체 및 이의 용도 | |
CN112807288B (zh) | 一种特异靶向感染部位的中性粒细胞膜仿生纳米材料的制备方法 | |
CN111249449B (zh) | 一种细胞外囊泡—白介素-10纳米靶向药物及其制备方法和应用 | |
DK2043697T3 (en) | ERYTHROCYTES CONTAINING MAGNETIC RESONANCE IMAGING CONTRACTORS | |
CN113355290B (zh) | 一种抗肿瘤的工程化外泌体、制备方法和应用 | |
CN112516109B (zh) | 一种基于间充质干细胞的融合癌细胞膜仿生纳米粒及其制备方法 | |
CN113616810A (zh) | 一种靶向p-选择素的工程化细胞外囊泡组合物及其制备方法和应用 | |
CN115960838A (zh) | 一种内外工程化外泌体及其制备方法与应用 | |
CN114224838B (zh) | 一种肿瘤微环境激活的融合膜包裹的仿生纳米递送***及其制备方法及应用 | |
CN114042147B (zh) | 靶向调控线粒体呼吸链的微纳水凝胶微球及其制备与应用 | |
US20220354802A1 (en) | Process of making membrane lipid coated nanoparticles | |
CN114949244A (zh) | 一种靶向改造的红细胞外泌体 | |
CN115581810B (zh) | 一种富含外泌体的水凝胶及其制备方法和应用 | |
WO2023226203A1 (zh) | 一种骨靶向细胞外囊泡及其制备方法和应用 | |
CN116515762A (zh) | 一种促进糖尿病创面愈合的工程化改造外泌体及其制备方法和应用 | |
EP3395829B1 (en) | Gelatin particles, method for producing gelatin particles, gelatin particle-containing cell, and method for producing gelatin particle-containing cell | |
CN112826942B (zh) | 靶向颅脑损伤病灶的脂质纳米递药***及其制备方法和应用 | |
Wang et al. | Preparation of genetically or chemically engineered exosomes and their therapeutic effects in bone regeneration and anti-inflammation | |
WO2023073609A1 (en) | Method for obtaining concentrated populations of extracellular vesicles washed of the physiopathological load thereof | |
CN117919205A (zh) | 一种表达cxcr4的仿生载药纳米粒子及其制备方法和应用 | |
Nutsch | Oral and transdermal delivery of branched amphiphilic peptide capsules in vivo | |
CN115590969A (zh) | 一种增强外泌体的细胞摄取效率的方法及应用 |
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 |