Lyophilization Based Isolation of Exosomes

被引:19
作者
Qazi, Rida e Maria [1 ]
Sajid, Zahra [1 ]
Zhao, Chunqiu [2 ]
Hussain, Irfan [1 ]
Iftikhar, Fizza [1 ]
Jameel, Muhammad [1 ]
Rehman, Fawad Ur [1 ]
Mian, Afsar Ali [1 ]
机构
[1] Aga Khan Univ, Ctr Regenerat Med & Stem Cells Res, 1st Flour, Juma Bldg, Stadium Rd, Karachi 74800, Pakistan
[2] Southeast Univ, State Key Lab Bioelect, Sipailou 2, Nanjing 210096, Peoples R China
基金
中国博士后科学基金;
关键词
exosomes; nano biopolymers; lyophilization; drug delivery; Ph plus leukemia; SELECTIVE INHIBITOR; FLUORESCENCE; DISEASE; POTENT; CELLS;
D O I
10.3390/ijms241310477
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Exosomes are nanoscale extracellular vesicles which regulate intercellular communication. They have great potential for application in nanomedicine. However, techniques for their isolation are limited by requirements for advanced instruments and costly reagents. In this study, we developed a lyophilization-based method for isolating exosomes from cultured cells. The isolated exosomes were characterized for protein content using Bradford assay, and for size distribution and shape using scanning electron microscopy (SEM) and nanoparticles tracking analysis (NTA). In addition, CD63, CD9, CD81, HSP70 and TSG101 were evaluated as essential exosomal surface markers using Western blot. Drug loading and release studies were performed to confirm their drug delivery properties using an in vitro model. Exosomes were also loaded with commercial dyes (Cy5, Eosin) for the evaluation of their drug delivery properties. All these characterizations confirmed successful exosome isolation with measurements of less than 150 nm, having a typical shape, and by expressing the known exosome surface protein markers. Finally, tyrosine kinase inhibitors (dasatinib and ponatinib) were loaded on the exosomes to evaluate their anticancer effects on leukemia cells (K562 and engineered Ba/F3-BCR-ABL) using MTT and Annexin-PI assays. The expression of MUC1 protein on the exosomes isolated from MCF-7 cells also indicated that their potential diagnostic properties were intact. In conclusion, we developed a new method for exosome isolation from cultured cells. These exosomes met all the essential requirements in terms of characterization, drug loading and release ability, and inhibition of proliferation and apoptosis induction in Ph+ leukemia cells. Based on these results, we are confident in presenting the lyophilization-based exosome isolation method as an alternative to traditional techniques for exosome isolation from cultured cells.
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页数:16
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